Serotonin receptor expression along the dorsal–ventral axis of mouse hippocampus

Using in situ hybridization, we describe, for the first time, the profiles of expression of serotonin receptors (Htr/5-HTR) along the dorsal–ventral axis of mouse hippocampus. cRNA probes for most Htrs, excluding Htr6, were used. All hippocampal subregions and the entorhinal cortex cells providing input into the hippocampus were examined. The study shows that some, but not all, Htrs are expressed in the cells of the hippocampal circuitry. At both the subfield and the cell type levels, a somewhat overlapping pattern is observed. Four serotonin receptors, Htr1a, Htr2a, Htr2c and Htr7, display an expression pattern that changes along the dorsal–ventral axis of the hippocampus. Given the proposed functional differentiation of the hippocampus along its long axis, with the dorsal pole more involved in cognitive functions and the ventral pole more involved in mood and anxiety, our results suggest that serotonin receptors enriched in the ventral pole probably contribute to mood- and anxiety-related behaviours.     

Physiological sites of deamidation and methyl esterification in sensory transducers of Halobacterium salinarum

In Halobacterium salinarum, up to 18 sensory transducers (Htrs) relay environmental stimuli to an intracellular signaling system to induce tactic responses. As known from the extensively studied enterobacterial system, sensory adaptation to persisting stimulus intensities involves reversible methylation of certain transducer glutamate residues, some of which originate from glutamine residues by deamidation. This study analyzes the in vivo deamidation and methylation of membrane-bound Htrs under physiological conditions. Electrospray ionization tandem mass spectrometry of chromatographically separated proteolytic peptides identified 19 methylation sites in 10 of the 12 predicted membrane-spanning Htrs. Matrix-assisted laser desorption/ionization mass spectrometry additionally detected three sites in two soluble Htrs. Sensory transducers contain a cytoplasmic coiled-coil region, composed of hydrophobic heptads, seven-residue repeats in which the first and the fourth residues are mostly hydrophobic. All identified Htr methylations occurred at glutamate residues at the second and/or third position of such heptads. In addition to singly methylated pairs of glutamate and/or glutamine residues, we identified singly methylated aspartate-glutamate and alanine-glutamate pairs and doubly methylated glutamate pairs. The largest methylatable regions detected in Htrs comprise six heptads along the coiled coil. One methylated glutamate residue was detected outside of such a region, in the signaling region of Htr14. Our analysis produced evidence supporting the predicted methyltransferase and methylesterase activities of halobacterial CheR and CheB, respectively. It furthermore demonstrated that CheB is required for Htr deamidations, at least at a specific glutamine-glutamate pair in Htr2 and a specific aspartate-glutamine pair in Htr4. Compared to previously reported methods, the described approach significantly facilitates the identification of physiological transducer modification sites.     

Late Maturation of Adult-Born Neurons in the Temporal Dentate Gyrus

Hippocampal function varies along its septotemporal axis, with the septal (dorsal) pole more frequently involved in spatial learning and memory and the temporal (ventral) pole playing a greater role in emotional behaviors. One feature that varies across these subregions is adult neurogenesis. New neurons are more numerous in the septal hippocampus but are more active in the temporal hippocampus during water maze training. However, many other aspects of adult neurogenesis remain unexplored in the context of septal versus temporal subregions. In addition, the dentate gyrus contains another functionally important anatomical division along the transverse axis, with the suprapyramidal blade showing greater experience-related activity than the infrapyramidal blade. Here we ask whether new neurons differ in their rates of survival and maturation along the septotemporal and transverse axes. We found that neurogenesis is initially higher in the infrapyramidal than suprapyramidal blade, but these cells are less likely to survive, resulting in similar densities of neurons in the two blades by four weeks. Across the septotemporal axis, neurogenesis was higher in septal than temporal pole, while the survival rate of new neurons did not differ. Maturation was assessed by immunostaining for the neuronal marker, NeuN, which increases in expression level with maturation, and for the immediate-early gene, Arc, which suggests a neuron is capable of undergoing activity-dependent synaptic plasticity. Maturation occurred approximately 1–2 weeks earlier in the septal pole than in the temporal pole. This suggests that septal neurons may contribute to function sooner; however, the prolonged maturation of new temporal neurons may endow them with a longer window of plasticity during which their functions could be distinct from those of the mature granule cell population. These data point to subregional differences in new neuron maturation and suggest that changes in neurogenesis could alter different hippocampus-dependent behaviors with different time courses.     

The CNS midline cells and spitz class genes are required for proper patterning of Drosophila ventral neuroectoderm.

The Drosophila embryonic central nervous system (CNS) develops from sets of neuroblasts (NBs) which segregate from the ventral neuroectoderm during early embryogenesis. It is not well established how each individual NB in the neuroectoderm acquires its characteristic identity along the dorsal-ventral axis. Since it is known that CNS midline cells and spitz class genes (pointed, rhomboid, single-minded, spitz and Star) are required for the proper patterning of ventral CNS and epidermis originated from the ventral neuroectoderm, this study was carried out to determine the functional roles of the CNS midline cells and spitz class genes in the fate determination of ventral NBs and formation of mature neurons and their axon pathways. Several molecular markers for the identified NBs, neurons, and axon pathways were employed to examine marker gene expression profile, cell lineage and axon pathway formation in the spitz class mutants. This analysis showed that the CNS midline cells specified by single-minded gene as well as spitz class genes are required for identity determination of a subset of ventral NBs and for formation of mature neurons and their axon pathways. This study suggests that the CNS midline cells and spitz class genes are necessary for proper patterning of the ventral neuroectoderm along the dorsal-ventral axis.     

Regulated vnd expression is required for both neural and glial specification in Drosophila.

The Drosophila embryonic CNS arises from the neuroectoderm, which is divided along the dorsal-ventral axis into two halves by specialized mesectodermal cells at the ventral midline. The neuroectoderm is in turn divided into three longitudinal stripes--ventral, intermediate, and lateral. The ventral nervous system defective, or vnd, homeobox gene is expressed from cellularization throughout early neural development in ventral neuroectodermal cells, neuroblasts, and ganglion mother cells, and later in an unrelated pattern in neurons. Here, in the context of the dorsal-ventral location of precursor cells, we reassess the vnd loss- and gain-of-function CNS phenotypes using cell specific markers. We find that over expression of vnd causes significantly more profound effects on CNS cell specification than vnd loss. The CNS defects seen in vnd mutants are partly caused by loss of progeny of ventral neuroblasts-the commissures are fused and the longitudinal connectives are aberrantly positioned close to the ventral midline. The commissural vnd phenotype is associated with defects in cells that arise from the mesectoderm, where the VUM neurons have pathfinding defects, the MP1 neurons are mis-specified, and the midline glia are reduced in number. vnd over expression results in the mis-specification of progeny arising from all regions of the neuroectoderm, including the ventral neuroblasts that normally express the gene. The CNS of embryos that over express vnd is highly disrupted, with weak longitudinal connectives that are placed too far from the ventral midline and severely reduced commissural formation. The commissural defects seen in vnd gain-of-function mutants correlate with midline glial defects, whereas the mislocalization of interneurons coincides with longitudinal glial mis-specification. Thus, Drosophila neural and glial specification requires that vnd expression by tightly regulated.     

Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina

Optic nerve formation requires precise retinal ganglion cell (RGC) axon pathfinding within the retina to the optic disc, the molecular basis of which is not well understood. At CNS targets, interactions between Eph receptor tyrosine kinases on RGC axons and ephrin ligands on target cells have been implicated in formation of topographic maps. However, studies in chick and mouse have shown that both Eph receptors and ephrins are also expressed within the retina itself, raising the possibility that this receptor-ligand family mediates aspects of retinal development. Here, we more fully document the presence of specific EphB receptors and B-ephrins in embryonic mouse retina and provide evidence that EphB receptors are involved in RGC axon pathfinding to the optic disc. We find that as RGC axons begin this pathfinding process, EphB receptors are uniformly expressed along the dorsal-ventral retinal axis. This is in contrast to the previously reported high ventral-low dorsal gradient of EphB receptors later in development when RGC axons map to CNS targets. We show that mice lacking both EphB2 and EphB3 receptor tyrosine kinases, but not each alone, exhibit increased frequency of RGC axon guidance errors to the optic disc. In these animals, major aspects of retinal development and cellular organization appear normal, as do the expression of other RGC guidance cues netrin, DCC, and L1. Unexpectedly, errors occur in dorsal but not ventral retina despite early uniform or later high ventral expression of EphB2 and EphB3. Furthermore, embryos lacking EphB3 and the kinase domain of EphB2 do not show increased errors, consistent with a guidance role for the EphB2 extracellular domain. Thus, while Eph kinase function is involved in RGC axon mapping in the brain, RGC axon pathfinding within the retina is partially mediated by EphB receptors acting in a kinase-independent manner.     

Independent quantitative trait loci influence ventral and dorsal hippocampal volume in recombinant inbred strains of mice

Anatomical and functional studies support segregation of the hippocampus into ventral and dorsal components along its septotemporal axis. However, it is unknown whether the development of these two components of the hippocampus is influenced by common or separate genetic factors. In this study, we used recombinant inbred strains of mice to determine whether the same or different quantitative trait loci (QTL) influence ventral and dorsal hippocampal volume. Using two sets of strains of recombinant inbred mice (BXD and AXB/BXA), we identified separate QTLs for ventral and dorsal hippocampal volume. In BXD mice, suggestive QTLs for ventral hippocampus were identified on chromosomes 2, 8 and 13, and a significant QTL for dorsal hippocampal volume was identified on chromosome 15. There was also a suggestive QTL for dorsal hippocampal volume on chromosome 13. In AXB/BXA mice, there were no significant or suggestive QTLs for ventral hippocampal volume, but a significant QTL for dorsal hippocampus was identified on chromosome 5. These findings suggest that the development of the ventral and dorsal components of the hippocampus is influenced by separate genetic loci.     

Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product

Drosophila females that lack Toll gene activity produce dorsalized embryos, in which all embryonic cells behave like the dorsal cells of the wild-type embryo. Injection of wild-type cytoplasm into young Toll- embryos restores their ability to produce a normal dorsal-ventral pattern in a position-dependent manner. No matter where the cytoplasm is injected relative to the dorsal-ventral axis of the egg shell, the position of the injected cytoplasm defines the ventralmost part of the rescued pattern. Although injection of wild-type cytoplasm into mutants at six other dorsal-group loci also restores the ability to produce lateral and ventral structures, only Toll- embryos lack any residual dorsal-ventral polarity. Experiments suggest that the activity of the Toll product is normally regulated by other dorsal-group genes and that the function of the Toll product is to provide the source for a morphogen gradient in the dorsal-ventral axis of the wild-type embryo.     

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/β-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.     

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.     

The Effects of Cholinergic and Dopaminergic Antagonists on Nicotine-Induced Cerebral Neurotransmitter Changes

In a continuing study of nicotine-induced mechanisms in brain areas associated with cognitive processes, the effects of cholinergic and dopaminergic antagonists on nicotine-induced changes in dopamine, norepinephrine, and serotonin were examined. These effects were measured via in vivo microdialysis in the dorsal and ventral hippocampus and in the prefrontal and medial temporal cortex of conscious, freely moving, adult male rats. Nicotine (0.3 mg/kg, free base) was administered subcutaneously and the antagonists were infused locally via the microdialysis probe. Nicotine alone induced an increase of dopamine and its metabolites in all areas, an increase of norepinephrine in the cortex, and an increase of the norepinephrine metabolite 4–hydroxy-3-methoxy-phenylglycol in all areas. Serotonin was decreased in the hippocampus and increased in the cortex. Nicotine-induced dopamine increases were inhibited by nicotinic (mecamylamine 100 μM, methyllycaconitine 500 μM), muscarinic (atropine 100 μM), and dopaminergic D1 (SCH23390 100 μM) and D2 (eticlopride 100 μM) antagonists, in the hippocampal and cortical areas. In the hippocampal areas, these antagonists had less significant effect on norepinephrine and serotonin. However, in the cortical areas, all antagonists inhibited the nicotine-induced increase of serotonin to varying degrees; and some, primarily nicotinic and dopamine D1 antagonists, inhibited the induced increase of norepinephrine. In the hippocampal and cortical areas, the mechanisms of nicotine-induced dopamine increase seem to be similar, but the mechanisms seem to be different for noradrenergic and serotonergic systems, as shown by the fact that nicotine induces no change in norepinephrine and a decrease in serotonin in the hippocampus, while it induces an increase in both in the cortex. Nicotine-induced dopamine release seems to be mediated, in part locally, by nicotinic and muscarinic receptors on dopaminergic cells. In contrast, nicotine’s effect on norepinephrine and serotonin is at least partially mediated by initial changes at other than local sites, and through different receptors. Thus, the effects of nicotine and the mechanisms involved differ for different neurotransmitters and in different brain areas.     

Differential Response of Hippocampal Subregions to Stress and Learning

The hippocampus has two functionally distinct subregions–the dorsal portion, primarily associated with spatial navigation, and the ventral portion, primarily associated with anxiety. In a prior study of chronic unpredictable stress (CUS) in rodents, we found that it selectively enhanced cellular plasticity in the dorsal hippocampal subregion while negatively impacting it in the ventral. In the present study, we determined whether this adaptive plasticity in the dorsal subregion would confer CUS rats an advantage in a spatial task–the radial arm water maze (RAWM). RAWM exposure is both stressful and requires spatial navigation, and therefore places demands simultaneously upon both hippocampal subregions. Therefore, we used Western blotting to investigate differential expression of plasticity-associated proteins (brain derived neurotrophic factor [BDNF], proBDNF and postsynaptic density-95 [PSD-95]) in the dorsal and ventral subregions following RAWM exposure. Lastly, we used unbiased stereology to compare the effects of CUS on proliferation, survival and neuronal differentiation of cells in the dorsal and ventral hippocampal subregions. We found that CUS and exposure to the RAWM both increased corticosterone, indicating that both are stressful; nevertheless, CUS animals had significantly better long-term spatial memory. We also observed a subregion-specific pattern of protein expression following RAWM, with proBDNF increased in the dorsal and decreased in the ventral subregion, while PSD-95 was selectively upregulated in the ventral. Finally, consistent with our previous study, we found that CUS most negatively affected neurogenesis in the ventral (compared to the dorsal) subregion. Taken together, our data support a dual role for the hippocampus in stressful experiences, with the more resilient dorsal portion undergoing adaptive plasticity (perhaps to facilitate escape from or neutralization of the stressor), and the ventral portion involved in affective responses.     

Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity.

Twelve maternal effect genes (the dorsal group and cactus) are required for the establishment of the embryonic dorsal-ventral axis in the Drosophila embryo. Embryonic dorsal-ventral polarity is defined within the perivitelline compartment surrounding the embryo by the ventral formation of a ligand for the Toll receptor. Here, by transplantation of perivitelline fluid we demonstrate the presence of three separate activities present in the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and sp?tzle embryos, respectively. These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventral formation of a ligand for the Toll protein. This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline space of wild-type embryos results in the formation of dorsalized embryos.